Birinapant sensitizes platinum-resistant carcinomas with high levels of cIAP to carboplatin therapy

Abstract

Platinum drugs are the frontline therapy in many carcinomas, including high-grade serous ovarian cancers. Clinically, high-grade serous carcinomas have an apparent complete response to carboplatin, but tumors invariably recur and response to platinum drugs diminishes over time. Standard of care prohibits re-administration of platinum drugs to these patients who are labeled as having platinum-resistant disease. In this stage patients are treated with non-platinum agents and outcomes are often poor. In vivo and in vitro data presented here demonstrate that this clinical dogma should be challenged. Platinum drugs can be an effective therapy even for platinum-resistant carcinomas as long as they are combined with an agent that specifically targets mechanisms of platinum resistance exploited by the therapy-resistant tumor subpopulations. High levels of cellular inhibitor of apoptosis proteins cIAP1 and 2 (cIAP) were detected in up to 50% of high-grade serous and non-high-grade serous platinum-resistant carcinomas. cIAP proteins can induce platinum resistance and they are effectively degraded with the drug birinapant. In platinum-resistant tumors with ≥22.4 ng of cIAP per 20 μg of tumor lysate, the combination of birinapant with carboplatin was effective in eliminating the cancer. Our findings provide a new personalized therapeutic option for patients with platinum-resistant carcinomas. The efficacy of birinapant in combination with carboplatin should be tested in high-grade serous carcinoma patients in a clinical trial.

Fig. 1

OS of mice bearing bioassay positive platinum-resistant or sensitive human HGSCs improved with birinapant and carboplatin co-administration. a Schema of in vitro organoid bioassay. Organoids treated with drug for 72 h were released, dissociated, and analyzed for survival by flow cytometry and passaging. Sensitivity to co-therapy was defined as ≤1% survival and no growth after passaging. b Graph showing the percentage of cells that survived treatment. Co-therapy sensitive lines S9-GODL and S1-GODL were eliminated, while co-therapy resistant lines S8-GODL and Ovcar-3 survived co-therapy. Results are mean ± SD, n = 3 replicates/cell line. c Experimental schema to assess the in vivo efficacy of birinapant and carboplatin co-therapy. Mice bearing IP human HGSCs were randomized to receive a 4-week course of vehicle, birinapant, carboplatin, or the combination of birinapant and carboplatin (n = minimum 8/arm). Mice were euthanized when they met NIH-defined endpoint criteria. d OS increased in mice bearing S9-GODL and S1-GODL tumors. (i) Co-therapy significantly increased OS in mice bearing platinum-resistant S9-GODL HGSC tumors (p < 0.0001, co-therapy vs. carboplatin). (ii) Co-therapy did not impact survival of mice bearing platinum-resistant S8-GODL tumors (p = 0.0753, co-therapy vs. carboplatin). (iii) Mice bearing platinum-sensitive S1-GODL tumors had increased OS with co-therapy (p = 0.01, co-therapy vs. carboplatin). (iv) Co-therapy did not improve OS in mice bearing Ovcar-3 tumors (p = 0.2340, co-therapy vs. carboplatin)

Fig. 2

Half of HGSC and non-HGSC carcinomas tested were sensitive to carboplatin and birinapant co-therapy. a Assessment of cell survival after drug treatment in the in vitro organoid bioassay (n = 3 replicates/specimen) revealed that in a cohort of 23 randomly selected primary patient HGSC specimens 13 were sensitive to co-therapy, while 10 were resistant. Single asterisk denotes samples clinically defined as platinum resistant. b Analysis of matched tumor specimens (three replicates/specimen) collected from spatially separated sites in the same patient (n = 6 patients) demonstrated that sensitivity to birinapant and carboplatin co-therapy was shared in multi-site HGSCs. c The response of platinum-resistant cell lines from cervical, bladder, lung, or colorectal cancers (n = 3 each) to co-therapy was examined in the in vitro organoid bioassay (n = 3 replicates/cell line). Half of the cell lines tested (CaSki, HeLa, J82, H226, SW620, and Colo205) were effectively targeted with co-therapy. In four of these cell lines (denoted by the symbol) no surviving cells could be detected by flow cytometry. In all six co-therapy sensitive lines, no growth was detected upon re-plating without drug. Results are mean ± SD

Fig. 3

Analysis of cIAP levels by western blot effectively segregated co-therapy sensitive vs. resistant HGSCs. a Schema for analysis of cIAP levels in HGSCs by semi-quantitative western blot and its correlation with co-therapy response. b cIAP protein levels were measured in 20 μg of lysate from the CA125 negative and positive HGSC populations and compared with a 40 ng standard of cIAP1 and 2 (20 ng each) included on each blot. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Levels of cIAP expression, quantified for each sample using ImageJ, were plotted against in vitro response to co-therapy. The expression of cIAP in the CA125 negative cells was significantly higher in co-therapy sensitive (>26.1 ng in 20 μg lysate) compared with co-therapy resistant (<26.1 ng in 20 μg lysate) HGSCs (p < 0.0001). c Levels of cIAP expression were determined in platinum-resistant non-HGSC carcinoma cell lines. Cells were treated for 72 h with vehicle (as control) or carboplatin (to enrich for platinum-resistant cells). Significantly higher cIAP expression was observed after carboplatin treatment in co-therapy sensitive compared with co-therapy resistant cell lines (threshold midpoint 37.5 ng cIAP in 20 μg cell lysate, threshold range 36.3–38.7 ng cIAP, p = 0.004). d Analysis of all platinum-resistant samples (HGSC and non-HGSC) demonstrates that cIAP levels in unfractionated or vehicle-treated tumor cells can segregate co-therapy sensitive (n = 9) vs. co-therapy resistant (n = 10) carcinomas in clinically defined platinum-resistant disease (threshold midpoint 22.4 ng cIAP in 20 μg cell lysate, threshold range 19.1–25.8 ng cIAP, 100% accuracy, p < 0.0001)

Fig. 4

Proposed strategy for overcoming platinum resistance in tumors of patients diagnosed with HGSC. a Schematic for a proposed clinical trial. We propose a proof of concept study aimed at determining the efficacy of birinapant and carboplatin co-therapy in improving survival of women with HGSC. Eligible subjects would be women with newly diagnosed or recurrent HGSC with co-therapy sensitive tumors based on the in vitro organoid bioassay. Patients would be treated with six cycles of carboplatin and concurrently receive the apoptosis-enhancing drug birinapant. Progression free and OS would be assessed and compared with historic controls. The validity of two biomarker assays, western blot and IHC, aimed at measuring levels and percentage of cIAP positive cells would be correlated with response to co-therapy. b Patients with platinum-resistant HGSCs are not re-treated with carboplatin in accordance with existing standards of care. At this point second-line chemotherapy agents are administered but clinical outcomes are often poor. We suggest a different approach that involves treating platinum-resistant HGSCs with carboplatin in combination with a pharmacologic agent (such as birinapant in tumors with high cIAP expression) that overcomes innate mechanisms of platinum resistance in CA125 negative platinum-resistant cells